High Throughput Screen

ABSTRACT

The present invention relates to a structure comprising a biological membrane and a porous or perforated substrate, a biological membrane, a substrate, a high throughput screen, methods for production of the structure membrane and substrate, and a method for screening a large number of test compounds in a short period. More particularly it relates to a structure comprising a biological membrane adhered to a porous or perforated substrate, a biological membrane capable of adhering with high resistance seals to a substrate such as perforated glass and the ability to form sheets having predominantly an ion channel or transporter of interest, a high throughput screen for determining the effect of test compounds on ion channel or transporter activity, methods for manufacture of the structure, membrane and substrate, and a method for monitoring ion channel or transporter activity in a membrane.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/875,860, filed May 2, 2013, which is a continuation of U.S.application Ser. No. 12/940,564, filed Nov. 5, 2010, now U.S. Pat. No.8,449,825, which is a continuation application of U.S. application Ser.No. 11/133,808, filed May 20, 2005, now U.S. Pat. No. 7,846,389, whichis a continuation of U.S. application Ser. No. 09/719,236, filed Apr.19, 2001, now U.S. Pat. No. 6,936,462, and claims priority of UKApplication No. 9812783.0, filed on Jun. 12, 1998, and InternationalApplication No. PCT/GB99/01871, filed Jun. 14, 1999, the disclosures ofwhich are incorporated fully herein by reference.

FIELD OF THE INVENTION

The present invention relates to a structure comprising a biologicalmembrane and a porous or perforated substrate, a biological membrane, asubstrate, a high throughput screen, methods for production of thestructure membrane and substrate, and a method for screening a largenumber of test compounds in a short period. More particularly it relatesto a structure comprising a biological membrane adhered to a porous orperforated substrate, a biological membrane capable of adhering withhigh resistance seals to a substrate such as perforated glass and theability to form sheets having predominantly an ion channel ortransporter of interest, a high throughput screen for determining theeffect of test compounds on ion channel or transporter activity, methodsfor manufacture of the structure, membrane and substrate, and a methodfor monitoring ion channel or transporter activity in a membrane.

BACKGROUND OF THE INVENTION

Ion channels are transmembrane proteins which form pores in the membranewhich allow ions to pass from one side to the other. Hille, B (ed).Ionic channels of excitable membranes. 1992. They may show ionspecificity, allowing specific ions to passively diffuse across amembrane down their electrochemical gradients. Although certain types ofchannels are on the average open all the time and at all physiologicalmembrane potentials (so-called leak channels), many channels have‘gates’ which open in response to a specific perturbation of themembrane. Perturbations known to cause opening of ion channels include achange in the electric potential across the membrane (voltage-gatedchannels), mechanical stimulation (mechanically-gated channels) or thebinding of a signalling molecule (ligand-gated channels).

Transporters are proteins in the cell membrane which catalyse themovement of inorganic ions such as Na⁺ and K⁺ as well as organicmolecules such as neurotransmitters as in the case of so-calledre-uptake pumps, e.g. GABA, dopamine and glycine. Two distinguishingfeatures of carriers versus pores are i) their kinetics-movement of ionsvia transporters is very much slower than the >10⁶ ions per second thatis encountered with ion channels and ii) ion channels conduct downelectrochemical gradients whereas transporters can ‘pump’ uphill i.e.against concentration gradients (Hille, 1992). The latter process isnormally directly dependent upon energy being provided in astoichiometric fashion.

Ion channel activity has been studied using a technique referred to as“patch clamping.” Hamill, O. P., Marty A., Neher, E., Sakmann, B. &Sigworth, F. J. (1981). Improved patch-clamp techniques forhigh-resolution current recording from cells and cell-free membranepatches. Pfluger's Archives, 391, 85-100. According to this technique asmall patch of cell membrane is generally isolated on the tip of amicropipette by pressing the tip against the membrane. It has beensuggested that if a tight seal between the micropipette and the patch ofmembrane is established electric current may pass through themicropipette only via ion channels in the patch of membrane. If this isachieved the activity of the ion channels and their effect on membranepotential, resistance and current may be monitored. If the electricpotential across the membrane remains constant the current supplied toit is equal to the current flowing through ion channels in the membrane.If ion channels in the membrane close, resistance of the membraneincreases. If the current applied remains constant the increase ofresistance is in direct proportion to an increase of electric potentialacross the membrane.

Many drugs are known to exert their effect by modulation of ionchannels, but the development of novel compounds acting on them ishampered considerably by the difficulty of screening at high-throughputrates for activity. Conventional electrophysio-logical methods such aspatch or voltage clamp techniques provide definitive mechanisticinformation but suffer from the problem that they are unsuited to therapid screening of test compounds.

WO96/13721 describes apparatus for carrying out a patch clamp techniqueutilized in studying the effect of certain materials on ion transferchannels in biological tissue. It discloses patch clamp apparatusutilizing an autosampler, such as those utilized with HPLC apparatus, toprovide a higher throughput than may be achieved by the conventionalpatch clamp technique. This apparatus suffers from the problems that itmerely semi-automates the drug delivery system, not the patch clamprecording. It therefore suffers from the same limitations as traditionalpatch-clamping with respect to speed of processing compounds and can inno way be considered a high-throughput system. The system still requireslinear processing (i.e. processing of data obtained for one cell afteranother). In direct contrast the invention described herein providesparallel processing and thus genuine high-throughput of compounds.

The term “biological membrane” used herein is taken to includeartificial membranes such as lipid bilayers and other membranes known toa person skilled in the art. Within the context of this specificationthe word “comprises” is taken to mean “includes” and is not intended tomean “is limited to only”.

SUMMARY OF THE INVENTION

The present invention relates to a structure comprising a biologicalmembrane and a porous or perforated substrate, a biological membrane, asubstrate, a high throughput screen, methods for production of thestructure membrane and substrate, and a method for screening a largenumber of test compounds in a short period. More particularly it relatesto a structure comprising a biological membrane adhered to a porous orperforated substrate, a biological membrane capable of adhering withhigh resistance seals to a substrate such as perforated glass and theability to form sheets having predominantly an ion channel ortransporter of interest, a high throughput screen for determining theeffect of test compounds on ion channel or transporter activity, methodsfor manufacture of the structure, membrane and substrate, and a methodfor monitoring ion channel or transporter activity in a membrane.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described by reference to the followingexamples of preferred embodiments and accompanying drawings in which:

FIGS. 1A and 1B shows an epithelial cell version of a screen accordingto an embodiment of the invention.

FIGS. 2A and 2B shows an embodiment of the screen of the inventionhaving a perforated substrate.

FIG. 3 shows adaption of a commercially available multi-well plate foruse in a screen according to an embodiment of the invention. The figureshows an integral multi-recording electrode head cluster.

FIG. 4 shows an embodiment using a movable recording head wherein asingle recording head reads single wells sequentially.

FIGS. 5A-5F shows an embodiment of a fluid matrix system wherein anarray of miniature recording chambers are created by dispensing dropletson to the recording substrate in a pre-determined pattern and density.FIG. 5 (f) shows the full sandwich (recording configuration) of thesystem.

FIGS. 6A-6D shows a further embodiment of a fluid matrix system whereinmultiple arrays of droplets are sandwiched together.

FIG. 7 shows a pore formed in a substrate, according to the invention.The light micrograph shows a pore in a thin glass substrate. The pore,which was approximately 2 micrometers in diameter, was manufactured byusing pulses of focused laser energy followed by a combination of firepolishing and plasma modification. The scale bar is 10 micrometersacross.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention addresses the problems associated with the knownscreens and screening methods. The invention has application principallyin the measurement of ion channel activity but also of transporterswhere these are electrogenic e.g. Na⁺/K⁺; Na⁺/Ca2⁺; glutamate re-uptaketransporter. Brew, H. & Attwell, D. (1987). Electrogenic glutamateuptake is a major current carrier in the membrane of axilotl retinalglial cells. Nature, 327, 707-9.

In the various embodiments shown in the figures, the followingcomponents are identified by the following reference numerals:

-   -   1: cells    -   2: permeabilized cell surface    -   3: voltage clamp    -   4: porous substrate    -   5: electrode    -   6: well wall    -   7: solution perfusion channel    -   8: cell line or primary cell    -   9: permeabilized cell    -   10: multiwell plate (e.g. 96 wells)    -   11: integral recording head cluster    -   12: multiplexer    -   13: ADC/computer    -   14: fluid level    -   15: pore    -   16: O-ring    -   17: recording assembly    -   18: cell plate    -   19: reference plate    -   20: recording plate    -   21: reference electrode    -   22: recording electrode    -   23: “demi-sandwich”    -   24: full sandwich (recording configuration)

In a first aspect the present invention provides a structure whichcomprises a biological membrane adhered with a high resistance seal to aporous or perforated substrate for use in a high through put screenwherein the biological membrane comprises an ion channel or transporter.

In a second aspect the invention provides a biological membrane for usein the structure which is capable of adhering to a substrate with a highresistance seal wherein each cell forms a tight junction with adjacentcells and expresses an ion channel which is localised in the cellmembrane.

In a third aspect the invention provides a substrate for use in a highthroughput screen which is perforated.

In a fourth aspect the invention provides a high throughput (HiT) screenfor the detection and assay of test compounds with activity on voltagegated ion channels which comprises the biological membrane.

In a fifth aspect the invention provides a method of manufacturing astructure comprising a biological membrane adhered with a highresistance seal to a perforated substrate which comprises the steps ofselecting a substrate, perforating it, introducing a biological membraneto the substrate and sealing each pore with biological membrane.

In a sixth aspect the invention provides a method of manufacturing thebiological membrane which comprises the steps of selecting a cell type,evaluating it for ability to form contiguous layers of cells with tightjunctions and for low to negligible numbers of voltage gated ionchannels, culturing the cells on a substrate and ensuring that acontiguous layer of cells is grown.

In a seventh aspect the invention provides a method of manufacturing aperforated substrate which comprises the steps of shining a laser ofpreselected focal area, power or time of exposure at a coverslip toperforate it. This method also may include the additional step ofmodification of the perforated area by exposure to plasma and/orlocalised heating in order to attain the appropriate level of smoothnessof the perforation(s).

In an eighth aspect the invention provides a method of screening for thedetection or assay of compounds with activity on ion channels whichcomprises the steps of placing a biological membrane which expresses ionchannels of interest in contact with test compound in physiologicalsolution or non-physiological solution comprising a solvent such asdimethyl sulphoxide and measuring the resistance or impedance of thebiological membrane under the influence of test compound.

Preferably an embodiment of the biological membrane comprises cellshaving an ion channel or transporter which naturally resides in the cellmembrane thereof, or it can be inserted by transfection with cDNA and/orcRNA encoding the ion channel or transporter. The invention thus has theadvantage that is permits studies of native channels or transporterswhere the precise subunit composition is unknown or indeed where themolecular identity is completely unknown (i.e. not yet cloned) but alsoheterologously-expressed cloned channels or transporters where theidentity of the native channel or transporter is known or where preciseknowledge is required of the interaction of compound structures andchemical moieties of the ion channel or transporter. Therefore thesystem is substantially more versatile then existing approaches whichare insensitive and rely on getting high concentrations of cells (notalways possible with neurones) and high signal to noise ratios whichlimits their use to only certain types of cells and ion channels.

Preferably an embodiment of the biological membrane comprises aplurality of ion channels or transporters which are predominantlypreselected ion channels or transporters of interest. This provides theinvention with the advantage of permitting parallel screening ofdifferent channels potentially providing an even higher throughput ofcompounds.

More preferably an embodiment of the biological membrane comprisesgenetically engineered cells which have been engineered to predominantlyexpress an ion channel or transporter.

Preferably the ion channels are voltage gated ion channels.

Preferably an embodiment of the biological membrane comprises cellsselected from the group which comprises HEK-293 cells, geneticallymodified Chinese hamster ovary (CHO) cells, primary neuronal tissue suchas hippocampus, dorsal root ganglia, superior cervical ganglia etc.;skeletal muscle; smooth muscle; cardiac muscle; immune cells; epithelia;endothelia etc.

CHO cells and CHO sub-clones such as CHO-KI and CHO-dhfr (also known asDukx) have exceptionally low levels of endogenous ion channel expressionthus providing the advantage of having excellent signal to noisecharacteristics within a mammalian cell environment. Similarly, HEK-293(human embryonic kidney) cells express low levels of native channels andprovide a human expression ‘background’. Both these expression systemsare infinitely preferable to the well-used Xenopus oocyte techniquewhere not only are native channels and subunits abundant, but theamphibian cell environment differs in important ways from mammaliancells.

Preferably an embodiment of the biological membrane comprises ionchannels having rapid activation and inactivation kinetics whichexisting methods of high-throughput screening cannot resolve. Existingsystems, therefore, average transient ion channel signals frequentlyover periods of many seconds. Channels inactivating with time-constantsof the order of milliseconds and without a steady-state presence areeffectively undetectable in such systems. The invention presented

here however, has the advantage that it can easily resolve such kineticsjust as traditional patch clamping does, but at high-throughput rates.

Preferably an embodiment of the biological membrane comprises ionchannels which show specificity for ions selected from the group whichcomprises sodium, potassium, calcium, chloride.

Preferably an embodiment of the biological membrane comprises acontiguous layer of cells capable of adhering with a high resistanceseal to substrates selected from the group which comprises perforatedglass, plastics, rubber, polytetraflurotethylene (PTFE), PTFE/glassfabric and polyethylene terephthalate (PETP).

Preferably an embodiment of the biological membrane comprises apseudo-epithelium wherein one face of a contiguous layer of cells ispermeabilized thereby providing access to the interior of the cells.This has the great advantage of providing the means for current andvoltage-clamping which is not possible with any existing high-throughputscreening system. Not only does this permit high time-resolutionrecording but it also provides the means to stimulate or activatevoltage-gated ion channels in a precise and controlled manner. Forexample, it is not necessary to alter the ionic composition e.g. byelevating K⁺ to depolarize cells, which in itself can modulate thekinetics of ion channels (e.g. K⁺ channels) and also obscure theactivity of novel ligands by competition at ion channel binding sites.This is a very great advantage over all existing systems.Permeabilization also allows the introduction to the cytosol ofcompounds that otherwise could not do so either by virtue of molecularweight or physicochemical characteristics.

Preferably an embodiment of the biological membrane comprises acontiguous layer of cells which is permeabilized by an antibioticselected from the group which comprises amphotericin and nystatin; ordetergent selected from the group which comprises digitonin and saponin;or physical disruption using a high voltage field; or by enzymaticdigestion of a part of the membrane using an appropriate enzyme.

An advantage of using high voltage fields to permeabilize the membrane(electropermeabilisation) is that such a technique can permeabilize theplasmamembrane while sparing smaller intracellular structures such asmitochondria and endoplasmic reticulum. The technique can also becontrolled very precisely and would not necessitate a facility toexchange solutions in a lower chamber of the recording apparatus.

Preferably an embodiment of the substrate comprises a perforatedcoverslip.

Preferably an embodiment of the substrate has pores of diameters between0.5Φm and 10Φm. More preferably the pores are of diameters between 1Φmand 7Φm. More preferably the diameter is 1-2Φm.

Preferably an embodiment of the substrate comprises a grid of pores ofgreater number than 4 but less than 10. This provides the advantage of astatistically acceptable number of parallel recordings (i.e. >4) in eachtreatment but small enough that the ratio of pores to cells can be madevanishingly small and thus the probability that a pore is sealed withand therefore occluded by a cell extremely high.

Preferably an embodiment of the substrate according to the invention ismanufactured of a material selected from the group which comprisesglass, plastics, rubber, polytetraflurotethylene (PTFE), PTFE/glassfabric and polyethylene terephthalate (PETP).

Preferably an embodiment of the screen comprises:

-   -   a plurality of chambers, each having a permeable peripheral        surface providing    -   a substrate for the biological membrane;    -   a plurality of wells each capable of receiving a chamber and a        test compound    -   in a physiological solution or non-physiological solution        comprising dimethyl sulfoxide (DMSO) or other solvent;    -   a plurality of reference electrodes, at least one having        electrical contact with each well;    -   a movable recording head carrying at least one recording        electrode thus providing the basic requirement for automated        recording of ion channel activity in a multiwell plate format;    -   means for measuring electrical resistance or impedance between        the recording and reference electrodes; wherein    -   electrical current may pass between the recording and reference        electrodes through the permeable peripheral surface of each        chamber only via ion channels or transporters in the biological        membrane.

Preferably an embodiment of the screen comprises wells which areprovided by a multiwell plate. The advantage of this being that highthroughput can be achieved using industry-standard components which canbe processed using commercially available automated equipment androbotics. Users will have the possibility of using their existing plateprocessing equipment thus containing costs in establishing astate-of-the-art high-throughput electrophysiology screen.

Preferably an embodiment of the screen comprises a perforated substratefor the biological membrane.

Preferably a further embodiment of the screen comprises a structure orbiological membrane described above having ion channels of interest inan array of droplets on a porous substrate. Preferably an array ofminiature recording chambers is created by placing a ‘lid’ incorporatingrecording electrodes over the matrix of droplets such that a meniscus ofthe droplet solution is established. Preferably a test compound inelectrically conducting solution is placed in at least one of thedroplets or applied via access ports in the ‘lid’ and theresistance/impedance (in current-clamp configuration) of the biologicalmembrane or conductance (in voltage-clamp configuration) is measuredunder the influence of the test compound. An advantage of this approachis that sheets of substrate can be designed without the need to transferpieces of substrate (e.g. discs) to multiwell plates and also obviatescomplex chamber design with seals, ‘O’-rings and the like. The inventioncan still accommodate addition of solutions and has an additionaladvantage of using very small volumes and thus small quantities ofreagents and cells. Excellent insulation is afforded by the air gapsbetween adjacent droplets.

Preferably an embodiment of the recording head comprises a singlerecording electrode capable of being moved to visit each chambersequentially. More preferably an embodiment of the recording headcomprises a plurality of recording electrodes arranged in a line. Evenmore preferably the recording head comprises a plurality of recordingelectrodes arranged in a matrix. The advantage of this configuration isthat simultaneous recording from all wells is possible via adata-acquisition multiplexing system.

Preferably an embodiment of the screen is capable of multiplexing up to384 recording elements to a data acquisition system utilizing multiplevoltage-clamp amplifiers. This has the advantage of providing extremelyhigh time resolution and effectively simultaneous measurement from allwells. This has the advantage of providing the TERM system with thepotential to achieve throughput rates similar to the best possible forconventional fluorescence-based ligand-receptor binding assays (≧150,000compounds per week).

Preferably an embodiment of the method of manufacturing the structurecomprises the steps of simultaneously perforating a coverslip andsealing the pores with biological membrane. This embodiment provides theadvantage of eliminating steps in the establishment of the finalconfiguration, namely procedures required to optimise the probability ofa cell sealing with pores in the perforated substrate. This has theadvantage of simplifying the final product.

Preferably an embodiment of the method of manufacturing the biologicalmembrane includes the step of permeabilizing one surface of thecontiguous layer of cells thereby providing access to the interior ofthe cells. This has the great advantage of providing the means forcurrent and voltage-clamping which is not possible with any existinghigh-throughput screening system. Not only does this permit hightime-resolution recording but is also provides the means to stimulate oractivate voltage-gated ion channels in a precise and controlled manner.For example, it is not necessary to alter the ionic composition e.g. byelevating K⁺ to depolarize cells, which in itself can modulate thekinetics of ion channels (e.g. K⁺ channels) and also obscure theactivity of novel ligands by competition at ion channel binding sites.This is a very great advantage over all existing systems.Permeabilization also allows the introduction to the cytosol ofcompounds that otherwise could not do so either by virtue of molecularweight or physicochemical characteristics.

Preferably the permeabilization is carried out by the step of contactingthe surface with an antibiotic selected from the group which comprisesamphotericin and nystatin; or detergent selected from the group whichcomprises digitonin and saponin; or physical disruption using a highvoltage field; or by enzymatic digestion of a part of the cell membraneusing an appropriate enzyme.

An advantage of using high voltage fields to permeabilize the membrane(electropermeabilisation) is that such a technique can permeabilize theplasmamembrane while sparing smaller intracellular structures such asmitochondria and endoplamic reticulum. The technique can also becontrolled very precisely and would not necessitate a facility toexchange solutions in a lower chamber of the recording apparatus.

Preferably an embodiment of the method of manufacturing the biologicalmembrane includes the steps of transfecting cells with cDNA or cRNAencoding an ion channel of interest and cloning cells expressing the ionchannel of interest. These steps provide the invention with theadvantage of permitting studies of heterologously expressed clonedchannels where the identity of the native channel is known or whereprecise knowledge is required of the interaction of compound structuresand chemical moieties of the ion channel.

Preferably an embodiment of the method of manufacturing the perforatedsubstrate comprises the steps of adjusting the profile, taper ordiameter of the pore with a laser.

Preferably the laser source is controlled by an automated stage undercontrol of a computer and inverted phase-contrast microscope whichprovides the advantage of permitting visual examination of the porecharacteristics e.g. profile, taper and diameter.

Preferably an embodiment of the method of manufacturing the perforatedsubstrate comprises other non-laser methods such as photo-etching,casting and physical piercing of the substrate.

Preferably an embodiment of the screening method comprises the step ofmeasuring ion channel activity by monitoring trans-epithelial resistancemeasurements (TERM) across an intact cell layer.

In a further embodiment of the screening method a surface of thecontiguous cell layer is preferably permeabilized thereby providingaccess to the interior of the cells. This has the great advantage ofproviding the means for current and voltage-clamping which is notpossible with any existing high-throughput screening system. Not onlydoes this permit high time-resolution recording but is also provides themeans to stimulate or activate voltage-gated ion channels in a preciseand controlled manner. For example, it is not necessary to alter theionic composition e.g. by elevating K⁺ to depolarize cells, which initself can modulate the kinetics of ion channels (e.g. K⁺ channels) andalso obscure the activity of novel ligands by competition at ion channelbinding sites. This is a very great advantage over all existing systems.Permeabilization also allows the introduction to the cytosol ofcompounds that otherwise could not do so either by virtue of molecularweight or physicochemical characteristics.

Preferably a surface of the contiguous cell layer is permeabilized byantibiotics selected from the group which comprises amphotericin andnystatin; or detergents selected from the group which comprisesdigitonin and saponin; or physical disruption using a high voltagefield; or by enzymatic digestion of a part of the membrane using anappropriate enzyme thereby permitting intracellular voltage or currentmeasurements to be made.

An advantage of using high voltage fields to permeabilize the membrane(electropermeabilisation) is that such a technique can permeabilize theplasmamembrane while sparing smaller intracellular structures such asmitochondria and endoplasmic reticulum. The technique can also becontrolled very precisely and does not necessitate a facility toexchange solutions in a lower chamber of the recording apparatus.

Preferably an embodiment of the invention provides a screening methodwhich includes the step of multiplexing up to 384 recording elements toa data acquisition system utilizing multiple voltage-clamp amplifiers.This has the advantage of providing extremely high time resolution andeffectively simultaneous measurement from all wells. This has theadvantage of providing the TERM system with the potential to achievethroughput rates similar to the best possible for conventionalfluorescence-based ligand-receptor binding assays (≧150,000 compoundsper week).

Preferably an embodiment of the method of screening for the detection orassay of compounds with activity on ion channels of interest in an arrayof droplets on a porous substrate. An array of miniature recordingchambers may be created by placing a ‘lid’ incorporating recordingelectrodes over the matrix of droplets such that a meniscus of dropletsolution is established. A test compound in conducting solution isplaced in at least one of the droplets or applied via access ports inthe ‘lid’ and the resistance of the biological membrane or conductance(in voltage-clamp configuration) is measured under the influence of thetest compound.

In an alternative embodiment of the screening method the biologicalmembrane is placed in a plurality of chambers and test compound inphysiological solution, or non-physiological solution comprising asolvent eg dimethyl sulphoxide, is added to the chambers.

Preferably an embodiment of the screening method comprises the steps ofdrug delivery and washing of the multi-well plate.

Preferably an embodiment of the screening method incorporates a step ofstimulation of cells involving the use of a photoactivatible ‘ionscavenger’ eg of ions such as K⁺. The active entity can be released byflashing the entire plate at once with a high intensity light source ega laser or Xenon lamp. The advantage of this system is that membranepotential can be altered by altering the ionic distribution in anon-invasive fashion and with fine temporal control.

It has surprisingly been found that a biological membrane can be adheredwith a high resistance seal to a perforated substrate for use in a highthroughput screen for test compounds having activity on ion channels.This was considered unobvious to a person skilled in the art at theoutset in view of the fact that achievement of a high resistance sealhas not been possible without an undue burden. Furthermore, perforatedsubstrates having a biological membrane sealed thereto have not beensuggested for use in high throughput screens.

It has surprisingly been found that a biological membrane capable ofadhering with a high resistance seal to a substrate may be constructedfor use in a high throughput screen. Surprisingly it has been found thatthe biological membrane may be constructed having ion channels which arepredominantly the ion channels of interest. Furthermore, it hassurprisingly been found that a high throughput screen may be constructedand used to detect and assay a throughput of test compounds which may bein excess of 30000 per week.

Surprisingly the screen may be used to obtain bona fide electrophysiological data relating to functional ion channel activity.

The biological membrane of the invention was unobvious at the outset toa person skilled in the art. Construction of a biological membranehaving high resistance seals with a substrate such as perforated glasshad not been achieved and was not considered possible without an undueburden. In addition construction of a membrane having ion channels whichare predominantly an ion channel of interest had not been consideredpossible without an undue burden.

The high throughput screens and methods of the invention were unobviousat the outset to a person skilled in the art in view of the fact that itwas not considered possible without an undue burden to screen the highthroughput of test compounds which may be achieved by the invention.

In addition to the advantage of a high-throughput of test compounds,embodiments of the screen and method of the invention may providefunctional assays (cf, say ligand binding) in which the mode of action(e.g. blocking or enhancing) of the test compound on voltage gated ionchannels is measured via changes in membrane resistance or by recordingthe current flowing through ion channels in the membrane directly.

EXAMPLES

An embodiment of the screen of the invention comprises a multi wellplate with integrated recording devices, by which means a massivelyparallel voltage clamp (MPVC) can be performed on a plurality of wellsin a plate within short periods of time (ca. 1-60 s). Preferablycommercially available 96 or 384 well plates are employed, or the 96 or384 position array format is adopted.

An embodiment of the screen of the invention preferably provides athroughput of test compounds in excess of 30,000 per week with bona fideelectrophysio-logical ‘read-out’ of functional ion channel activity. Anembodiment of the screen may provide high resolution both in terms oftime; for a 96 well plate, 1 ms/point/voltage clamp setup. Thesensitivity to modulation of channel activity is ≧1%.

An embodiment of the present invention provides a method which comprisesthe steps of measuring transepithelial electrical resistance across acontiguous layer of cells expressing predominantly an ion channel ofinterest. It will be apparent to a person skilled in the art that themethod depends on adherence of the cells to a substrate that allowsformation of tight junctions between adjacent cells such that ahigh-resistance sheet is formed. Changes in activity of the ion channelsexpressed in the membranes of individual cells are reflected by changesin the resistance across the sheet as a whole. In a refinement of thisembodiment of the invention, access to the interior of the cellscomprising the sheet is obtained by means which permit populationcurrent and voltage clamp recording to be made.

Transepithelial resistance measurements have been carried out asfollows:

a) Epithelial/Endothelial Type Cells.

The overall transepithelial resistance is composed of two principalcomponents, the sum of the ‘whole-cell’ conductances of the individualcells making-up the pseudo-epithelium and the sum of the intercellularconductance pathways.

Naturally-occurring epithelial (and endothelial) cells form tightjunctions with one another. This tight packing reduces the leakage ofions/solutes between cells, resulting in relatively low numbers ofintercellular conductance pathways across the membrane as a whole. Thus,where conditions permit tight junctions to form, changes in the celltransmembrane conductance are measurable. One approach to this methodwas to select a host cell with appropriate growth properties. Cell-typeswhich also express native ion channels at low levels are consideredsuitable for expression of cloned ion channels. A large hurdle for aperson skilled in the art in this approach lay in obtaining cells inwhich the latter is true.

b) Transepithelial Resistance Measurement in Non-Epithelial Cells.

An alternative to the approach described above is to use non-epithelialcells that are known to express negligible numbers of ion channels oftheir own as a basic expression system for cloned cells which expression channels of choice. There are several cell-types that fulfill thiscriterion. However, a large hurdle to a person skilled in the art waspresented in that they do not form contiguous layers of cells in culturewith tight junctions. In an embodiment of the invention there isprovided a high resistance ‘epithelial’ layer in which this hurdle hasbeen overcome.

Transepithelial Current Measurement (Massively Parallel Voltage Clamp(MPVC)

An embodiment of the invention was obtained by gaining access to theinterior of cells in the ‘epithelium’ by disrupting or permeabilizingone face of each cell contributing to the cell layer. This has beencarried out by chemical methods, for example by allowing certain typesof antibiotics (e.g. amphotericin) or detergents (digitonin) to comeinto contact with one face of the cell surface or through physicaldisruption e.g. using high voltage fields (electroporation/integralzapper).

Electrical Recording Systems

A number of systems have been developed and they are outlined below:

Pilot Test Systems

For pilot testing of the integrity of pseudo-epithelial layers,transepithelial resistance was measured using a chamber into whichpermeable tissue culture supports were inserted (FIGS. 1 and 2). Cellswere grown in culture to confluency on this support. In the case ofperforated substrates, the material (e.g. coverslip) was inserted in apurpose-built test rig which permitted variation in the pressure in thelower compartment and/or upper chamber and at the same time allowedresistance measurements to be made (FIGS. 1 a and 2 a). To avoidpolarization of the electrodes, an oscillating voltage signal was passedacross the cell-layer via linear or circular electrodes positioned aboveand below the permeable support on which the layer of cells was growingand the impedance of the cell-layer was measured. In the case ofpermeabilized cell-layers (FIGS. 1 b and 2 b), voltage and current-clamprecording was carried out using a voltage-clamp amplifier interfacedwith a computer to control experimental protocols and sample the data.

Scale-Ups and Operational Systems

In either TERM or MPVC commercial screens utilize a multiwell platemeasuring system (FIG. 3) or equivalent (e.g. using a droplet matrixgenerated using a nano litre piezo-dispenser). This was derived to someextent from the pilot test rig but required the design of an integralrecording head of which embodiments of the invention include a number ofpossibilities. They are described below.

-   -   i) single recording head which reads single wells sequentially        (FIG. 4).    -   ii) moveable linear row of recording heads (e.g. 12 for a 96        well plate system; 24 for a 384 well system) which are moved        across the plate in 8 (96 well) or 16 (384 well) steps.    -   iii) electrode matrix built into the plate with multiplexing for        recording headstage & acquisition system. For larger density        plates multiple voltage-clamps were used to maintain sampling        frequency and therefore time resolution (FIG. 3).    -   iv) droplet system (FIG. 5).

Multiwell Plate Adaptation

Embodiments of the screen of the present invention preferably include anintegral automated pipettor in the working versions of TERM and MPVC.

Preferably embodiments of the screen of the invention include a facilityfor washing recording heads between use in separate rows.

According to an embodiment of the invention the method of manufacture ofthe biological membrane comprises the steps of obtaining a highresistance seal with perforated glass substrate (or other support)and/or the step of obtaining a cell-line having the ability to formsheets and having low or negligible numbers of native ion channels.

Epithelial Cell Approach

Naturally occurring cell-lines and engineered cell-lines have beendeveloped. They are described below.

Naturally-Occurring Cell-Lines

Cell-lines referred to in the literature have been evaluated for ‘offthe shelf suitability. Initial candidates included ECV-304, RBE4 and C6glioma cells. Criteria for use were:

a) ability to form contiguous layers of cells with tight junctions;transepithelial resistance of 3125Σcm⁻².

b) low to negligible numbers of background voltage-gated ion channels asassessed by whole cell patch clamp by standard methods. Preferably theconductance level is #2 nS per cell.

Engineered Cell-Lines

A suitable cell-line may be prepared by molecular engineering. Ifbackground conductances of the naturally-occurring cell-lines were abovethe threshold given in (b) above, the cell-line was assessed forpossible gene knock-out to engineer a novel host cell.

Artificial Epithelia Perforated Substrates

Perforated substrates have been developed as set out below:

Laser-Generated Substrates a) Prototypes

Glass coverslips were perforated in a sequential fashion (1 hole at atime) using a laser energy source in conjunction with automated stageunder computer control and an inverted optics microscope. This permittedprototypes to be constructed with fine control of parameters such asfocal area, laser power and time of exposure. The ability to achievehigh resistance sealing between cells and substrate was tested usingcoverslips created in this way.

Grid patterns were reproducibly generated in variable formats by meansof a computer-controlled stage which was interfaced with the laser viathe computer. Coverslips of various materials including glass (as wellas plastics and other biocompatible materials) were used. Theirdiameters were 1 Omm (96 well plate) or 5 mm (384 well plate); and ofvariable thickness (ca. 1-20 μm). Pores were generated with diametersvarying between 0.5μ and 10μ. The profile of the pore, its taper andinternal and external diameters were evaluated to optimise sealing withthe test cells. It is important to establish the appropriate level ofsmoothness of the pore. Pore density was optimized for signal-to-noisecharacteristics, fidelity of voltage-clamp recording and statisticalconsiderations.

To encourage sealing between cell and pore, a number of approaches weretaken (FIG. 1). They are outlined below:

i) negative pressure in lower liquid compartment e.g. using a venturieffect caused by flowing solution across the ventral orifice and/or bysupplying the flowing solution at a reduced overall pressure.

ii) positive pressure in the upper liquid compartment

iii) coating of the coverslip with anti-adhesion material that is burnedoff in the pore region during the pore manufacturing process (i.e. laserinduced pore formation)

iv) facility to jog or vibrate coverslip to encourage cells to ‘find’pores before adhering to the substrate at non-pore locations

v) either a coverslip carousel or multiwell plate carousel to permitcentrifugation.

vi) application of voltage field across pores to move cells into poremouth.

Surprisingly, laser induced pore formation provided remarkable results.FIG. 7 shows a typical pore produced by this method. When physiologicalsolutions were added to either side of the pore, trans-substrateresistances, typically in the range 200 kOhms to 400 kOhms, wereroutinely observed. With the addition of cells, the observed resistancewas approximately double this figure. With the additional application ofone or more of the approaches outlined above, resistance measurementsapproaching the gigaohm range were observed.

b) Scale-Ups

Bulk perforation and simultaneous recording (sealing) were evaluated.The approach comprised ‘flashing’ the whole bottom surface of amultiwell plate (or equivalent matrix) with a high energy laser source.With appropriate well structure, the precise location of the requiredpores was known and with appropriate titration of cell density, a highprobability of a having a cell ‘in residence’ was achieved. The platewas perforated and the ventral cell surface breached almostsimultaneously. This required a much higher energy laser than that usedin protoypes (above).

c) Cell-Types

Although Chinese hamster ovary (CHO) cells have been used to develop theinvention, it will be apparent to a person of ordinary skill in the artthat a wide variety of cell-lines and primary cells such as neuronesisolated from intact tissues may be employed.

Other Perforation Methods

Alternative methods of perforating glass coverslips and other materialshave been evaluated such as etching, casting glass or plastics sheets.

Porous Rubber

Porous rubber substrates are commercially available for growing cells incell-culture. The porosity has been evaluated in the context of theresistance and current-measuring applications described herein.

Other Materials

It will be apparent to a person skilled in the art that additionalmaterials such as PTFE, PETP etc. may be employed in accordance with thepresent invention. These have the advantage of having high dielectricconstants but also of being manufactured in extremely thin sheets. Thishas the advantage of reducing the minimum series resistance in the wholesystem and also facilitating the introduction of exogenous substances tothe cell cytosol.

Multi-Well Plate Recording Apparatus

The basal multi-well plate recording apparatus preferably accommodates a96-well/location format. Preferably multiples of the 96-well format areconstructed with a minimal expansion to 384 well format. An advantage ofincreasing well-density is that the amount of test compound used isreduced and fewer plates are used with concomitant reductions inancillary running costs per compound tested.

The following two approaches have been evaluated:—

a) A TERM workstation designed to interface with commercially-availablerobots and plate processors.

b) Fully integrated stand-alone system which provides plate handling,solution changes and recording headstages.

Fluid Matrix System

An array of miniature recording chambers were created by dispensingdroplets containing a suspension of cells onto the recording substratein a predetermined pattern and density (see FIG. 5). The substrate canbe of any of the types described above e.g. perforated glass, plastic,rubber, etc. The complete recording configuration is accomplished byplacing a ‘lid’, or moveable recording head, incorporating recordingelectrodes over the matrix of droplets such that a meniscus of thedroplet solution is established as exemplified in FIG. 5. An array ofdroplets may also be generated on the reverse of the porous substrate toprovide a conducting pathway to at least one reference electrode andalso the means by which substances may be applied to the lower surfaceof the substrate and hence cell membranes. Similarly, reagents can beapplied via a further droplet matrix applied to the ‘recording plate’ asshown in FIG. 6. Drug solutions may be dispensed onto a recording “head”plate; the plate may then be inverted; and the inverted plate may thenbe docked with the cell matrix to bring the drug into contact with thecells. The advantage of this approach is that sheets of substrate can bedesigned without the need to transfer substrate discs to multiwellplates and also obviates complex chamber design with seals, ‘O’-ringsand the like. The invention can still accommodate addition of solutionsand has the additional advantage of using very small volumes and thussmall quantities of reagents and cells.

What is claimed is:
 1. A method of screening for the detection or assayof compounds with activity on ion channels or transporters, comprising:placing a biological cellular membrane in contact with a pore in aporous substrate, the pore having a diameter between 0.5 μm and 10 μm;flowing a solution containing at least one said compound from an inletto an outlet through a perfusion channel disposed below the poroussubstrate, said channel communicating with said pore and said biologicalcellular membrane; and performing patch clamping measurement across thebiological cellular membrane between an positioned electrode below thesubstrate in contact with said flowing solution and an electrodepositioned above the substrate electrically communicating with thebiological cellular membrane.
 2. The method of screening of claim 1,wherein the substrate is disposed in a well of a multi-well plate. 3.The method of screening of claim 1, further comprising adhering saidbiological cellular membrane to said porous substrate over said porewith a seal between said membrane and said porous substrate havingsufficient electrical resistance to facilitate said patch clampingmeasurements.
 4. The method of screening of claim 3, wherein saidadhering forms a high resistance seal.
 5. The method of screening ofclaim 3, further comprising: applying a pressure differential acrosssaid porous substrate; and forming a lower pressure in the solutionperfusion channel as compared to above the substrate.
 6. The method ofscreening of claim 1, wherein said at least one pore has a diameterbetween 1 and 7 μm.
 7. The method of screening of claim 6, wherein saidat least one pore has a diameter of 1-2 μm
 8. The method of screening ofclaim 1, further comprising said pore is with a laser.
 9. The method ofscreening of claim 8, further comprising selecting the porous substratefrom a material comprising one of glass, PTFE or PTEP.
 10. The method ofscreening of claim 1, further comprising: connecting a voltage-clampamplifier connected between said electrodes; and configuring saidvoltage-clamp amplifier for patch clamping measurements.
 11. A method ofmanufacturing a structure for screening for the detection or assay ofcompounds with activity on ion channels or transporters, comprising:selecting a substrate; perforating the substrate with plural poreshaving a diameter between 0.5 μm and 10 μm; smoothing the pore edges toprovide a shape and smoothness appropriate to permit adherence of abiological cellular membrane with a high resistance seal thereto;positioning said substrate along a bottom of a multi-well plate forminga bottom wall thereof with at least one pore disposed in each well. 12.The method of claim 11, wherein said selecting comprises selecting amaterial from a group which comprises glass, plastics, rubber,polytetraflurotethylene (PTFE), PTFE/glass fabric and polyethyleneterephthalate (PETP).
 13. The method of claim 11, wherein saidperforating comprises one of laser forming, photo-etching, casting andphysical piercing.
 14. The Method of claim 13, wherein said laserforming comprises computer controlled forming of a grid of pores on saidsubstrate with plural pores in each said well.
 15. The method of claim13, further comprising, subsequent to said perforating, exposing thesubstrate to a localized heat or electrical plasma to increase thesmoothness of the pores.
 16. An apparatus for patch clampingmeasurements to detect and assay compounds with ion channel activity ina biological cellular membrane, comprising: a plate defining a pluralityof wells configured to electrically communicate with an electrode of avoltage-clamp amplifier, said plate being configured and dimensioned tobe positioned above a solution perfusion channel with flow of fluidthrough said perfusion channel in contact with a bottom side of saidwells; and a porous substrate disposed in a bottom of each said well,wherein said substrate is formed of a material permitting adherence of abiological cellular membrane on an upper side thereof with a highresistance seal between the membrane and said porous substrate havingsufficient electrical resistance to facilitate patch clampingmeasurements across a biological membrane adhered to said substrate,said substrate defines plural pores in each said well, and said poreshave a diameter between 1 μm and 7 μm, a smoothness sufficient to formsaid high resistance seal, and are disposed in said well forcommunication with fluid flowing in the solution perfusion channel. 17.The apparatus of claim 16, wherein said pores are laser formed.
 18. Theapparatus of claim 16, further comprising a structure for supportingsaid plate and defining said solution perfusion channel having an inletand an outlet for flow of fluid containing said compounds therethroughbelow said plate in contact with said substrate.
 19. The apparatus ofclaim 18, further comprising at least a first electrode disposable ineach said well, and at least a second electrode disposed in saidsolution perfusion channel in electrical communication with said firstelectrode across the porous substrate.
 20. The apparatus of claim 19,further comprising a voltage-clamp amplifier connected between saidelectrodes and configured for patch clamping measurements.